Human Zven polypeptides

ABSTRACT

The present invention provides polypeptides and compositions comprising amino acid residues 23 to 108 of SEQ ID NO: 2, polypeptides and compositions comprising amino acid residues 28 to 108 of SEQ ID NO: 2, including affinity tags and the like, useful for stimulating gastrointestinal contractility, gastric emptying, intestinal transit, and treating gastroparesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/982,168, filed Nov. 5, 2004, now U.S. Pat. No. 7,122,349, which is acontinuation of U.S. application Ser. No. 10/212,355, filed Aug. 2,2002, now U.S. Pat. No. 6,828,425, which is a divisional of U.S.application Ser. No. 09/712,529, filed Nov. 14, 2000, now U.S. Pat. No.6,485,938, which claims the benefit of U.S. Provisional Application No.60/165,905, filed Nov. 16, 1999, U.S. Provisional Application No.60/184,875, filed Feb. 25, 2000, U.S. Provisional Application No.60/197,750, filed April 19, 2000, and U.S. Provisional Application No.60/210,332, filed Jun. 7, 2000, all of which are herein incorporated byreference.

TECHNICAL FIELD

The present invention relates generally to new polypeptides havingdiagnostic and therapeutic uses. In particular, the present inventionrelates to polypeptides, designated “Zven1” and “Zven2,” and to nucleicacid molecules encoding the Zven polypeptides.

BACKGROUND OF THE INVENTION

Cellular differentiation of multicellular organisms is controlled byhormones and polypeptide growth factors. These diffusable moleculesallow cells to communicate with each other, to act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

Hormones and growth factors influence cellular metabolism by binding toreceptor proteins. Certain receptors are integral membrane proteins thatbind with the hormone or growth factor outside the cell, and that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of receptors are soluble intracellular molecules.

Wnt proteins are emerging as one of the pre-eminent families ofsignaling molecules in animal development. To date, murine Wnt genesinclude Wnt-1, Wnt-2, Wnt-2B/13, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-5B,Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-10A, Wnt-10B, Wnt-11, andWnt-15, while the following human Wnt genes have been described: Wnt-1,Wnt-2, Wnt-2B/13, Wnt-3, Wnt-4, Wnt-5A, Wnt-7A, Wnt-8A, Wnt-8B, Wnt-10B,Wnt-11, Wnt-14, and Wnt-15. See, for example, Nusse and Varmus, Cell31:99 (1982), van Ooyen et al., EMBO J. 4:2905 (1985), ainwright et al.,EMBO J. 7:1743 (1988), McMahon and McMahon, Development 107:643 (1989),Gavin et al., Genes Dev. 4:2319 (1990), Roelink et al., Proc. Nat'lAcad. Sci. USA 87:4519 (1990), Roelink and Nusse, Genes Dev. 5:381(1991), Clark et al., Genomics 18:249 (1993), Roelink et al., Genomics17:790 (1993), Adamson et al., Genomics 24:9 (1994), Huguet et al.,Cancer Res. 54:2615 (1994), Bouillet, Mech. Dev. 58:141 (1996), Ikegawaet al., Cytogenet. Cell Genet. 74:149 (1996), Katoh et al., Oncogene13:873 (1996), Lako et al., Genomics 35:386 (1996), Wang andShackleford, Oncogene 13:1537 (1996), Bergstein, Genomics 46:450 (1997),Bui et al., Oncogene 14:1249 (1997), and Grove et al., Development125:2315 (1998).

Wnt genes typically encode secreted glycoproteins having 350-400 aminoacids, and the proteins often include a conserved pattern of 23-24cysteine residues in addition to other invariant residues (Cadigan andNusse, Genes & Dev. 11:3286 (1997)). Following cellular secretion, Wntproteins are believed to reside mainly in the extracellular matrix or toassociated with the cellular surface.

According to the classical Wnt signaling pathway model, Wnt proteinsinduce gene expression by de-repressing a signal pathway via a so-called“Frizzled” transmembrane receptor (see, for example, Brown and Moon,Curr. Opin. Cell Biol. 10:182 (1998)). In the absence of Wnt, glycogensynthase kinase-3β activity results in the degradation of the freecytosolic pool of β-catenin. The association of cognate Wnt proteins andFrizzled receptors leads to the activation of a signaling pathway. Themost proximal intracellular component of this pathway is the Disheveledprotein, which becomes phosphorylated and inhibits glycogen synthasekinase-3β. Consequently, the pool of intracellular β-catenin increases,and β-catenin can interact with members of the lymphoid enhancer/T cellfactor (LEF/TCF) family of architectural transcription factors in thenucleus. These complexes bind consensus LEF/TCF sites in promoters andinduce transcription of Wnt-responsive genes.

The Wnt proteins are multipotent, and the proteins are capable ofinducing different biological responses in both embryonic and adultcontexts (see, for example, Ingham, TIG 12:382 (1996)). This type ofbroad activity is shared with fibroblast growth factors, transforminggrowth factors β, and nerve growth factors (Nusse and Varmus, Cell69:1073 (1992)). When over-expressed, Wnt proteins can promote tumorformation (Erdreich-Epstein and Shackleford, Growth Factors 15:149(1998)). Knock-out mutations in mice have shown Wnt proteins to beessential for brain development, and the out growth of embryonicprimordia for kidney, tail bud and limb bud (McMahon and Bradley, Cell62:1073 (1990), Thomas and Capecchi, Nature 346:847 (1990), Stark etal., Nature 372:679 (1994), Takada et al., Genes Dev. 8:174 (1994), andParr and McMahon, Nature 374:350 (1995)).

Several secreted factors inhibit Wnt signaling (see, for example, Finchet al., Proc. Nat'l Acad. Sci. USA 94:6770 (1997); Moon et al., Cell88:725 (1997); Luyten et al., WO 98/16641); Brown and Moon, Curr. Opin.Cell Biol. 10:182 (1998); Aikawa et al., J. Cell. Sci. 112:3815 (1999)).The Frzb proteins, for example, bind to secreted Wnt proteins andprevent productive interactions between Wnt and Frizzled proteins. Theseproteins contain a region that is homologous to a putative Wnt-bindingdomain of Frizzled proteins. Wnt-inhibitory factor-1 is another type ofsecreted protein, which binds to Wnt proteins and inhibits Wnt signaling(Hsieh et al., Nature 398:431 (1999)). Wnt-inhibitory factor-1 proteinsare produced by fish, amphibia, and mammals, indicating the importanceof these inhibitory proteins (Hsieh et al., Nature 398:431 (1999)).

Inhibitors of Wnt signaling can be used to block the inducement of tumorformation by inappropriate Wnt expression. Accordingly, a need existsfor the provision of new Wnt inhibitory proteins.

BRIEF SUMMARY OF THE INVENTION

The present invention provides members of a new human gene family,designated as “Zven,” and, in particular, illustrative members of thegene family, designated “Zven1” and “Zven2.” The present invention alsoprovides Zven1 and Zven2 polypeptides and fusion proteins, nucleic acidmolecules encoding such polypeptides and proteins, and methods for usingthese nucleotide and amino acid sequences.

DESCRIPTION OF THE INVENTION

1. Overview

The present invention provides nucleic acid molecules that encode humanZven polypeptides. An illustrative nucleic acid molecule containing asequence that encodes the Zven1 polypeptide has the nucleotide sequenceof SEQ ID NO:1. The encoded polypeptide has the following amino acidsequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI TGACDKDSQC GGGMCCAVSIWVKSIRICTP MGKLGDSCHP LTRKVPFFGR RMHHTCPCLP GLACLRTSFN RFICLAQK (SEQ IDNO:2). Thus, the Zven1 nucleotide sequence described herein encodes apolypeptide of 108 amino acids. The putative signal sequences of Zven1polypeptide reside at amino acid residues 1 to 20, 1 to 21, and 1 to 22of SEQ ID NO:2.

Zven1 is expressed in eosinophils, and northern analysis indicates Zven1gene expression is present in human testicular tissue and peripheralblood lymphocytes. As described in Example 1, Zven1 is expressed in Bcell, T cell, monocyte, and granulocyte cell lines. Moreover, Zven1 geneexpression was detectable in unactivated monocytes, but not in activatedmonocytes. Thus, Zven1 gene expression can be used to differentiatebetween unactivated and activated monocytes. Example 2 describesstudies, which indicate that Zven1 can inhibit the proliferation of lungtumor cells. The Zven1 gene resides in human chromosome 3p21.1-3p14.3.

An illustrative nucleic acid molecule containing a sequence that encodesthe Zven2 polypeptide has the nucleotide sequence of SEQ ID NO:4. Theencoded polypeptide has the following amino acid sequence: MRGATRVSIMLLLVTVSDCA VITGACERDV QCGAGTCCAI SLWLRGLRMC TPLGREGEEC HPGSHKVPFFRKRKHHTCPC LPNLLCSRFP DGRYRCSMDL KNINF (SEQ ID NO:5). Thus, the Zven2nucleotide sequence described herein encodes a polypeptide of 105 aminoacids. The putative signal sequences of Zven2 polypeptide reside atamino acid residues 1 to 17, and 1 to 19 of SEQ ID NO:5.

Northern analyses show that the Zven2 gene is highly expressed in humantesticular and ovarian tissue. High levels of Zven2 gene expression werealso detected in placenta, adrenal gland, and prostate. In contrast,little or no Zven2 gene expression was evident in heart, brain, lung,small intestine, liver, skeletal muscle, kidney, pancreas, spleen,thymus, colon, peripheral blood lymphocytes, stomach, thyroid, spinalcord, lymph node, trachea, and bone marrow. Accordingly, Zven2 nucleicacid probes and anti-Zven2 antibodies can be used to differentiatebetween various tissues.

Sequence analysis revealed a homology relationship between Zven2 anddkk-1, a potent inhibitor of Wnt action reported in amphibians andhumans (Glinka et al., Nature 391:357 (1998); Fedi et al., J. Biol.Chem. 274:19465 (1999)). Since the activation of Wnt signaling cancontribute to the neoplastic process, a Wnt inhibitor can provide auseful therapeutic protein.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% identical toamino acid residues 23 to 108 of SEQ ID NO:2. Certain of such isolatedpolypeptides can specifically bind with an antibody that specificallybinds with a polypeptide consisting of the amino acid sequence of SEQ IDNO:2. Particular polypeptides can inhibit the proliferation of lungtumor cells. An illustrative polypeptide is a polypeptide that comprisesthe amino acid sequence of SEQ ID NO:2.

Similarly, the present invention includes provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% identical toamino acid residues 20 to 105 of SEQ ID NO:5, wherein such isolatedpolypeptides can specifically bind with an antibody that specificallybinds with a polypeptide consisting of the amino acid sequence of SEQ IDNO:5. An illustrative polypeptide is a polypeptide that comprises theamino acid sequence of SEQ ID NO:5.

The present invention also provides polypeptides comprising an aminoacid sequence selected from the group consisting of: (1) amino acidresidues 21 to 108 of SEQ ID NO:2, (2) amino acid residues 22 to 108 ofSEQ ID NO:2, (3) amino acid residues 23 to 108 of SEQ ID NO:2, (4) aminoacid residues 82 to 108 of SEQ ID NO:2, (5) amino acid residues 1 to 78(amide) of SEQ ID NO:2, (6) amino acid residues 1 to 79 of SEQ ID NO:2,(7) amino acid residues 21 to 78 (amide) of SEQ ID NO:2, (8) amino acidresidues 21 to 79 of SEQ ID NO:2, (9) amino acid residues 22 to 78(amide) of SEQ ID NO: 2, (10) amino acid residues 22 to 79 of SEQ ID NO:2, (11) amino acid residues 23 to 78 (amide) of SEQ ID NO:2, (12) aminoacid residues 23 to 79 of SEQ ID NO:2, (13) amino acid residues 20 to108 of SEQ ID NO:2, (14) amino acid residues 20 to 72 of SEQ ID NO:2,(15) amino acid residues 20 to 79 of SEQ ID NO:2, (16) amino acidresidues 20 to 79 (amide) of SEQ ID NO:2, (17) amino acid residues 21 to72 of SEQ ID NO:2, (18) amino acid residues 21 to 79 (amide) of SEQ IDNO: 2, (19) amino acid residues 22 to 72 of SEQ ID NO: 2, (20) aminoacid residues 22 to 79 (amide) of SEQ ID NO:2, (21) amino acid residues23 to 72 of SEQ ID NO:2, (22) amino acid residues 23 to 79 (amide) ofSEQ ID NO:2, (23) amino acid residues 28 to 108 of SEQ ID NO:2, (24)amino acid residues 28 to 72 of SEQ ID NO: 2, (25) amino acid residues28 to 79 of SEQ ID NO:2, (26) amino acid residues 28 to 79 (amide) ofSEQ ID NO:2, (27) amino acid residues 75 to 108 of SEQ ID NO:2, (28)amino acid residues 75 to 79 of SEQ ID NO:2, and (29) amino acidresidues 75 to 78 (amide) of SEQ ID NO:2. Illustrative polypeptidesconsist of amino acid sequences (1) to (29).

The present invention further includes polypeptides comprising an aminoacid sequence selected from the group consisting of: (a) amino acidresidues 20 to 105 of SEQ ID NO:5, (b) amino acid residues 18 to 105 ofSEQ ID NO:5, (c) amino acid residues 1 to 70 of SEQ ID NO:5, (d) aminoacid residues 20 to 70 of SEQ ID NO:5, (e) amino acid residues 18 to 70of SEQ ID NO:5, (f) amino acid residues 76 to 105 of SEQ ID NO:5, (g)amino acid residues 66 to 105 of SEQ ID NO:5, and (h) amino acidresidues 82 to 105 of SEQ ID NO:5. Illustrative polypeptides consist ofamino acid sequences (a) to (h).

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention also includes anti-idiotype antibodies that specifically bindwith such antibodies or antibody fragments. The present inventionfurther includes compositions comprising a carrier and a peptide,polypeptide, antibody, or anti-idiotype antibody described herein.

The present invention also provides isolated nucleic acid molecules thatencode a Zven polypeptide, wherein the nucleic acid molecule is selectedfrom the group consisting of (a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule encodingthe amino acid sequence of SEQ ID NO:2, (c) a nucleic acid molecule thatremains hybridized following stringent wash conditions to a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 161of SEQ ID NO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, or to the complement of the nucleotide sequence of eithernucleotides 66 to 161 of SEQ ID NO:1 or nucleotides 288 to 389 of SEQ IDNO:1, (d) a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO:6, (e) a nucleic acid molecule encoding the amino acidsequence of SEQ ID NO:5, (f) a nucleic acid molecule that remainshybridized following stringent wash conditions to a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 334 to 405of SEQ ID NO:4, or to the complement of the nucleotide sequence ofnucleotides 334 to 405 of SEQ ID NO:4.

Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by the nucleic acidmolecule and the corresponding amino acid sequence of either SEQ ID NO:2or SEQ ID NO:5 is due to a conservative amino acid substitution. Thepresent invention further contemplates isolated nucleic acid moleculesthat comprise a nucleotide sequence of nucleotides 132 to 389 of SEQ IDNO:1, and nucleotides 148 to 405 of SEQ ID NO:4.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells comprising these vectors and expression vectors.Illustrative host cells include bacterial, yeast, avian, fungal, insect,mammalian, and plant cells. Recombinant host cells comprising suchexpression vectors can be used to prepare Zven polypeptides by culturingsuch recombinant host cells that comprise the expression vector and thatproduce the Zven protein, and, optionally, isolating the Zven proteinfrom the cultured recombinant host cells. The present invention furtherincludes products made by such processes.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors.

The present invention also contemplates methods for detecting thepresence of Zven1 RNA in a biological sample, comprising the steps of(a) contacting a Zven1 nucleic acid probe under hybridizing conditionswith either (i) test RNA molecules isolated from the biological sample,or (ii) nucleic acid molecules synthesized from the isolated RNAmolecules, wherein the probe has a nucleotide sequence comprising aportion of the nucleotide sequence of SEQ ID NO:1, or its complement,and (b) detecting the formation of hybrids of the nucleic acid probe andeither the test RNA molecules or the synthesized nucleic acid molecules,wherein the presence of the hybrids indicates the presence of Zven1 RNAin the biological sample. Analogous methods can be used to detect thepresence of Zven2 RNA in a biological sample, wherein the probe has anucleotide sequence comprising a portion of the nucleotide sequence ofSEQ ID NO:4, or its complement.

The present invention further provides methods for detecting thepresence of Zven polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide eitherconsisting of the amino acid sequence of SEQ ID NO:2 or consisting ofthe amino acid sequence of SEQ ID NO:5, wherein the contacting isperformed under conditions that allow the binding of the antibody orantibody fragment to the biological sample, and (b) detecting any of thebound antibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold.

Illustrative biological samples include human tissue, such as an autopsysample, a biopsy sample, and the like.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zven1 gene expression maycomprise a container that comprises a nucleic acid molecule, wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides66 to 161 of SEQ ID NO:1, (b) a nucleic acid molecule comprising thenucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1, (c) anucleic acid molecule comprising the complement of the nucleotidesequence of nucleic acid molecules (a) or (b), (d) a nucleic acidmolecule that is a fragment of (a) consisting of at least eightnucleotides, (e) a nucleic acid molecule that is a fragment of (b)consisting of at least eight nucleotides, and (f) a nucleic acidmolecule that is a fragment of (c) consisting of at least eightnucleotides. A kit for detection of Zven2 gene expression may comprise acontainer that comprises a nucleic acid molecule, wherein the nucleicacid molecule is selected from the group consisting of (a) a nucleicacid molecule comprising the nucleotide sequence of nucleotides 334 to405 of SEQ ID NO:4, (b) a nucleic acid molecule comprising thecomplement of the nucleotide sequence of (a), (c) a nucleic acidmolecule that is a fragment of (a) consisting of at least eightnucleotides, and (d) a nucleic acid molecule that is a fragment of (b)consisting of at least eight nucleotides. Such kits may also comprise asecond container that comprises one or more reagents capable ofindicating the presence of the nucleic acid molecule.

On the other hand, a kit for detection of Zven protein may comprise acontainer that comprises an antibody, or an antibody fragment, thatspecifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 or consisting of the amino acid sequence of SEQID NO:5.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQID NO:5.

The present invention further provides variant Zven1 polypeptides, whichcomprise an amino acid sequence that shares an identity with the aminoacid sequence of SEQ ID NO:2 selected from the group consisting of atleast 70% identity, at least 80% identity, at least 90% identity, atleast 95% identity, or greater than 95% identity, and wherein anydifference between the amino acid sequence of the variant polypeptideand the amino acid sequence of SEQ ID NO:2 is due to one or moreconservative amino acid substitutions. Illustrative variant Zven2polypeptides, which comprise an amino acid sequence that shares anidentity with the amino acid sequence of SEQ ID NO:5 selected from thegroup consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,and wherein any difference between the amino acid sequence of thevariant polypeptide and the amino acid sequence of SEQ ID NO:5 is due toone or more conservative amino acid substitutions.

The present invention also provides fusion proteins comprising a Zven1polypeptide moiety or a Zven2 polypeptide moiety. Such fusion proteinscan further comprise an immunoglobulin moiety. A suitable immunoglobulinmoiety is an immunoglobulin heavy chain constant region, such as a humanFc fragment. The present invention also includes isolated nucleic acidmolecules that encode such fusion proteins.

The present invention also includes methods of inhibiting theproliferation of tumor cells (e.g., lung tumor cells), comprising thestep of administering a composition comprising Zven1 to the tumor cells.In an in vivo approach, the composition is a pharmaceutical composition,administered in a therapeutically effective amount to a subject, whichhas a tumor. Such in vivo administration can provide at least onephysiological effect selected from the group consisting of decreasednumber of tumor cells, decreased metastasis, decreased size of a solidtumor, and increased necrosis of a tumor.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SPI, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces aZven1 or Zven2 peptide or polypeptide from an expression vector. Incontrast, such polypeptides can be produced by a cell that is a “naturalsource” of Zven1 or Zven2, and that lacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zven1 or Zven2polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zven1or Zven2 using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zven1or anti-Zven2 antibody, and thus, an anti-idiotype antibody mimics anepitope of Zven1 or Zven2.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zven1 monoclonal antibody fragment bindswith an epitope of Zven1.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide, which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zven1” or a “Zven1anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zven1 gene, or(b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zven1 gene. Similarly, an “anti-sense oligonucleotidespecific for Zven2” or a “Zven2 anti-sense oligonucleotide” is anoligonucleotide having a sequence (a) capable of forming a stabletriplex with a portion of the Zven2 gene, or (b) capable of forming astable duplex with a portion of an mRNA transcript of the Zven2 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zven1 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zven1 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zven1 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zven1 gene can be identified bydetermining whether the gene hybridizes with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, or its complement, understringent conditions. Similarly, a variant Zven2 gene and a variantZven2 polypeptide can be identified with reference to SEQ ID NO:4 andSEQ ID NO:5, respectively.

Alternatively, variant Zven genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zven1gene or variant Zven1 polypeptide, a variant gene or polypeptide encodedby a variant gene may be characterized by its ability to bindspecifically to an anti-Zven1 antibody. Similarly, a variant Zven2 geneproduct or variant Zven2 polypeptide may be characterized by its abilityto bind specifically to an anti-Zven2 antibody.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zven1 and Zven2genes. Within the context of this invention, a “functional fragment” ofa Zven1 (or Zven2) gene refers to a nucleic acid molecule that encodes aportion of a Zven1 (or Zven2) polypeptide, which specifically binds withan anti-Zven1 (anti-Zven2) antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to 110%.

3. Production of Human Zven1 and Zven2 Genes

Nucleic acid molecules encoding a human Zven1 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. Similarly, nucleic acid molecules encoding ahuman Zven2 gene can be obtained by screening a human cDNA or genomiclibrary using polynucleotide probes based upon SEQ ID NO:4. Thesetechniques are standard and well-established.

As an illustration, a nucleic acid molecule that encodes a human Zven1gene can be isolated from a human cDNA library. In this case, the firststep would be to prepare the cDNA library by isolating RNA from tissues,such as testis or peripheral blood lymphocytes, using methods well-knownto those of skill in the art. In general, RNA isolation techniques mustprovide a method for breaking cells, a means of inhibitingRNase-directed degradation of RNA, and a method of separating RNA fromDNA, protein, and polysaccharide contaminants. For example, total RNAcan be isolated by freezing tissue in liquid nitrogen, grinding thefrozen tissue with a mortar and pestle to lyse the cells, extracting theground tissue with a solution of phenol/chloroform to remove proteins,and separating RNA from the remaining impurities by selectiveprecipitation with lithium chloride (see, for example, Ausubel et al.(eds.), Short Protocols in Molecular Biology, 3^(rd) Edition, pages 4-1to 4-6 (John Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al., Methodsin Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [“Wu(1997)”]). Alternatively, total RNA can be isolated from tissue byextracting ground tissue with guanidinium isothiocyanate, extractingwith organic solvents, and separating RNA from contaminants usingdifferential centrifugation (see, for example, Chirgwin et al.,Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997)at pages 33-41).

In order to construct a cDNA library, poly(A) RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. 1, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A human genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Nucleic acid molecules that encode a human Zven1 or Zven2 gene can alsobe obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences described herein. General methods for screeninglibraries with PCR are provided by, for example, Yu et al., “Use of thePolymerase Chain Reaction to Screen Phage Libraries,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317-337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO: 1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-Zven antibodies, produced as described below, can also be used toisolate DNA sequences that encode human Zven genes from cDNA libraries.For example, the antibodies can be used to screen λgt11 expressionlibraries, or the antibodies can be used for immunoscreening followinghybrid selection and translation (see, for example, Ausubel (1995) atpages 6-12 to 6-16; Margolis et al., “Screening λ expression librarieswith antibody and protein probes,” in DNA Cloning 2: Expression Systems,2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press1995)).

As an alternative, a Zven gene can be obtained by synthesizing nucleicacid molecules using mutually priming long oligonucleotides and thenucleotide sequences described herein (see, for example, Ausubel (1995)at pages 8-8 to 8-9). Established techniques using the polymerase chainreaction provide the ability to synthesize DNA molecules at least twokilobases in length (Adang et al., Plant Molec. Biol. 21:1131 (1993),Bambot et al., PCR Methods and Applications 2:266 (1993), Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Ann. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zven cDNA or Zven genomic fragment can be determinedusing standard methods. Zven polynucleotide sequences disclosed hereincan also be used as probes or primers to clone 5′ non-coding regions ofa Zven gene. Promoter elements from a Zven gene can be used to directthe expression of heterologous genes in tissues of, for example,transgenic animals or patients treated with gene therapy. Theidentification of genomic fragments containing a Zven promoter orregulatory element can be achieved using well-established techniques,such as deletion analysis (see, generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zvenproteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zven gene in a cell is altered byintroducing into the Zven locus a DNA construct comprising at least atargeting sequence, a regulatory sequence, an exon, and an unpairedsplice donor site. The targeting sequence is a Zven 5′ non-codingsequence that permits homologous recombination of the construct with theendogenous Zven locus, whereby the sequences within the construct becomeoperably linked with the endogenous Zven coding sequence. In this way,an endogenous Zven promoter can be replaced or supplemented with otherregulatory sequences to provide enhanced, tissue-specific, or otherwiseregulated expression.

4. Production of Zven Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, which encode the Zven polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NOs:3and 6 are a degenerate nucleotide sequences that encompasses all nucleicacid molecules that encode the Zven polypeptides of SEQ ID NOs:2 and 5,respectively. Those skilled in the art will recognize that thedegenerate sequence of SEQ ID NO:3 also provides all RNA sequencesencoding SEQ ID NO:2, by substituting U for T, while the degeneratesequence of SEQ ID NO:6 also provides all RNA sequences encoding SEQ IDNO:5, by substituting U for T. Thus, the present invention contemplatesZven1 polypeptide-encoding nucleic acid molecules comprising nucleotide66 to nucleotide 389 of SEQ ID NO:1, and their RNA equivalents, as wellas Zven2 polypeptide-encoding nucleic acid molecules comprisingnucleotide 91 to nucleotide 405 of SEQ ID NO:4, and their RNAequivalents.

Table 1 sets forth the one-letter codes used within SEQ ID NOs:3 and 6to denote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs:3 and 6, encompassing allpossible codons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter • TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NOs:2 and 5. Variant sequences can be readily testedfor functionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed in SEQ ID NOs:3 and 6 serve astemplates for optimizing expression of polynucleotides in various celltypes and species commonly used in the art and disclosed herein.Sequences containing preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zven polypeptides fromother mammalian species, including porcine, ovine, bovine, canine,feline, equine, and other primate polypeptides. Orthologs of human Zvencan be cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses Zven. Suitable sources of mRNA can be identified byprobing northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue orcell line.

A Zven-encoding cDNA molecule can then be isolated by a variety ofmethods, such as by probing with a complete or partial human cDNA orwith one or more sets of degenerate probes based on the disclosedsequences. A cDNA can also be cloned using the polymerase chain reactionwith primers designed from the representative human Zven sequencesdisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to Zven polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOs:1 and 4 represent single alleles of human Zven1 and Zven2,respectively, and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the nucleotide sequencesshown in SEQ ID NOs:1 and 4, including those containing silent mutationsand those in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of SEQ ID NOs:2 and 5. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the Zvenpolypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules consisting of the nucleotide sequence of SEQ IDNO:1, to nucleic acid molecules consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, to nucleic acid moleculesconsisting of the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, to nucleic acid molecules consisting of the nucleotide sequenceof SEQ ID NO:4, to nucleic acid molecules consisting of the nucleotidesequence of nucleotides 334 to 405 of SEQ ID NO:4, or to nucleic acidmolecules consisting of nucleotide sequences that are the complements ofsuch sequences. In general, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions that influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM-1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zven1polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution (100× Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher temperature, such as about 65° C., in a solution that does notcontain formamide. Moreover, premixed hybridization solutions areavailable (e.g., EXPRESSHYB Hybridization Solution from CLONTECHLaboratories, Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. For example, nucleic acid moleculesencoding particular variant Zven1 polypeptides can remain hybridizedwith a nucleic acid molecule consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO: 1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, or their complements, followingwashing under stringent washing conditions, in which the wash stringencyis equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C., including0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65° C. In asimilar manner, nucleic acid molecules encoding particular Zven2variants can remain hybridized with a nucleic acid molecule consistingof the nucleotide sequence of nucleotides 334 to 405 of SEQ ID NO:4, orits complement, following washing under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C., including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1%SDS at 65° C. One of skill in the art can readily devise equivalentconditions, for example, by substituting SSPE for SSC in the washsolution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. As an illustration, nucleic acid molecules encoding particularvariant Zven1 polypeptides can remain hybridized with a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 161of SEQ ID NO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, or their complements, following washing under highly stringentwashing conditions, in which the wash stringency is equivalent to0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1% SDSat 50° C., or 0.2×SSC with 0.1% SDS at 65° C. Similarly, nucleic acidmolecules encoding particular Zven2 variants remain hybridized with anucleic acid molecule consisting of the nucleotide sequence ofnucleotides 334 to 405 of SEQ ID NO:4, or its complement, followingwashing under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated Zven1 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having 85%,90%, 95% or greater than 95% sequence identity to the sequences shown inSEQ ID NO:2, or their orthologs. Similarly, the present inventionprovides isolated Zven2 polypeptides having 85%, 90%, 95% or greaterthan 95% sequence identity to the sequences shown in SEQ ID NO:5, ortheir orthologs.

The present invention also contemplates Zven variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NOs:2 or 5, and a hybridization assay, as describedabove. For example, certain Zven1 gene variants include nucleic acidmolecules (1) that remain hybridized with a nucleic acid moleculeconsisting of the nucleotide sequence of nucleotides 66 to 161 of SEQ IDNO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1,or their complements, following washing under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., and (2) that encode a polypeptide having85%, 90%, 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:2. Alternatively, certain Zven1 variant genes canbe characterized as nucleic acid molecules (1) that remain hybridizedwith a nucleic acid molecule consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, or their complements, followingwashing under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and(2) that encode a polypeptide having 85%, 90%, 95% or greater than 95%sequence identity to the amino acid sequence of SEQ ID NO:2.

Moreover, certain Zven2 gene variants include nucleic acid molecules (1)that remain hybridized with a nucleic acid molecule consisting of thenucleotide sequence of nucleotides 334 to 405 of SEQ ID NO:4, or itscomplement, following washing under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C., and (2) that encode a polypeptide having 85%, 90%, 95% orgreater than 95% sequence identity to the amino acid sequence of SEQ IDNO:5. Alternatively, certain Zven2 variant genes can be characterized asnucleic acid molecules (1) that remain hybridized with a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 334 to 405of SEQ ID NO:4, or its complement, following washing under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) that encode apolypeptide having 85%, 90%, 95% or greater than 95% sequence identityto the amino acid sequence of SEQ ID NO:5.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZven1 or Zven2 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, and most preferably, three. The other parameters canbe set as: gap opening penalty=10, and gap extension penalty=1.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with theamino acid sequence of SEQ ID NOs:2 or 5. That is, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNOs:2 or 5, in which an alkyl amino acid is substituted for an alkylamino acid in a Zven1 or Zven2 amino acid sequence, an aromatic aminoacid is substituted for an aromatic amino acid in a Zven1 or Zven2 aminoacid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a Zven1 or Zven2 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a Zven1 or Zven2 amino acid sequence, an acidic amino acidis substituted for an acidic amino acid in a Zven1 or Zven2 amino acidsequence, a basic amino acid is substituted for a basic amino acid in aZven1 or Zven2 amino acid sequence, or a dibasic monocarboxylic aminoacid is substituted for a dibasic monocarboxylic amino acid in a Zven1or Zven2 amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zven1 or Zven2 are characterized by having atleast 70%, at least 80%, at least 85%, at least 90%, at least 95% orgreater than 95% sequence identity to a corresponding amino acidsequence disclosed herein (i.e., SEQ ID NO:2 or SEQ ID NO:5), whereinthe variation in amino acid sequence is due to one or more conservativeamino acid substitutions.

Conservative amino acid changes in a Zven1 gene and a Zven2 gene can beintroduced by substituting nucleotides for the nucleotides recited inSEQ ID NO:1 and SEQ ID NO:4, respectively. Such “conservative aminoacid” variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel (1995) at pages8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)).

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zven amino acidresidues.

Amino acid sequence analysis indicates that Zven1 and Zven2 shareseveral motifs. For example, one motif is “AVITGAC[DE][KR]D,” (SEQ IDNO:8), wherein acceptable amino acids for a given position are indicatedwithin square brackets. This motif occurs in Zven1 at amino acidresidues 28 to 37 of SEQ ID NO:2, and in Zven2 at amino acid residues 20to 29 of SEQ ID NO:5. Another motif is“CHP[GL][ST][HR]KVPFFX[KR]RXHHTCPCLP,” (SEQ ID NO:9), wherein acceptableamino acids for a given position are indicated within square brackets,and “X” can be any amino acid residue. This motif occurs in Zven1 atamino acid residues 68 to 90 in SEQ ID NO:2, and in Zven2 at amino acidresidues 60 to 82 of SEQ ID NO:5. The present invention includespeptides and polypeptides comprising these motifs.

Sequence analysis also indicated that Zven1 and Zven2 include variousconservative amino acid substitutions with respect to each other.Accordingly, particular Zven1 variants can be designed by modifying itssequence to include one or more amino acid substitutions correspondingwith the Zven2 sequence, while particular Zven2 variants can be designedby modifying its sequence to include one or more amino acidsubstitutions corresponding with the Zven1 sequence. Such variants canbe constructed using Table 4, which presents exemplary conservativeamino acid substitutions found in Zven1 and Zven2. Although Zven1 andZven2 variants can be designed with any number of amino acidsubstitutions, certain variants will include at least about X amino acidsubstitutions, wherein X is selected from the group consisting of 2, 5,7, 10, 12, 14, 16, 18, and 20.

TABLE 4 Zven1 Zven2 Amino acid Position Amino acid Position (SEQ ID NO:2) Amino acid (SEQ ID NO: 5) Amino acid 4 Leu 4 Ala 7 Ala 7 Val 9 Leu 9Ile 14 Leu 14 Val 35 Asp 27 Glu 36 Lys 28 Arg 42 Gly 34 Ala 48 Val 40Ile 50 Ile 42 Leu 52 Val 44 Leu 53 Lys 45 Arg 55 Ile 47 Leu 63 Lys 55Arg 66 Asp 58 Glu 71 Leu 63 Gly 72 Thr 64 Ser 73 Arg 65 His 80 Arg 72Lys 93 Ala 85 Leu 102 Phe 94 Tyr

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity, such as the ability to bind to an antibody, to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699 (1996).

The location of Zven1 or Zven2 receptor binding domains can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992), andWlodaver et al., FEBS Lett. 309:59 (1992). Moreover, Zven1 or Zven2labeled with biotin or FITC can be used for expression cloning of Zven1or Zven2 receptors.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed Zven1 or Zven2 nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zven1 or anti-Zven2 antibodies, can be recoveredfrom the host cells and rapidly sequenced using modern equipment. Thesemethods allow the rapid determination of the importance of individualamino acid residues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zven1 orZven2 polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zven1 or Zven2 polypeptide. As an illustration, DNA moleculeshaving the nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for the ability to bindanti-Zven antibodies. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zven gene can be synthesized using thepolymerase chain reaction.

Methods for identifying functional domains are well-known to those ofskill in the art. For example, studies on the truncation at either orboth termini of interferons have been summarized by Horisberger and DiMarco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques forfunctional analysis of proteins are described by, for example, Treuteret al., Molec. Gen. Genet. 240:113 (1993), Content et al., “Expressionand preliminary deletion analysis of the 42 kDa 2-5A synthetase inducedby human interferon,” in Biological Interferon Systems, Proceedings ofISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72(Nijhoff 1987), Herschman, “The EGF Receptor,” in Control of Animal CellProliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (AcademicPress 1985), Coumailleau et al., J. Biol. Chem. 270:29270 (1995);Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al.,Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant Molec.Biol. 30:1 (1996).

The present invention also contemplates functional fragments of a Zven1or Zven2 gene that have amino acid changes, compared with the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:5. A variant Zven gene can beidentified on the basis of structure by determining the level ofidentity with the particular nucleotide and amino acid sequencesdisclosed herein. An alternative approach to identifying a variant geneon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant Zven1 or Zven2 gene can hybridizeto a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1or SEQ ID NO:4, as discussed above.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zven1 or Zven2 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of SEQ ID NOs:2 or 5. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting a Zven1 orZven2 polypeptide, or by chemical peptide synthesis, as describedherein. Moreover, epitopes can be selected by phage display of randompeptide libraries (see, for example, Lane and Stephen, Curr. Opin.Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616(1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

Regardless of the particular nucleotide sequence of a variant Zven1 orZven2 gene, the gene encodes a polypeptide that may be characterized byits ability to bind specifically to an anti-Zven1 or anti-Zven2antibody.

In addition to the uses described above, polynucleotides andpolypeptides of the present invention are useful as educational tools inlaboratory practicum kits for courses related to genetics and molecularbiology, protein chemistry, and antibody production and analysis. Due toits unique polynucleotide and polypeptide sequences, molecules of Zven1or Zven2 can be used as standards or as “unknowns” for testing purposes.For example, Zven1 or Zven2 polynucleotides can be used as an aid, suchas, for example, to teach a student how to prepare expression constructsfor bacterial, viral, or mammalian expression, including fusionconstructs, wherein Zven1 or Zven2 is the gene to be expressed; fordetermining the restriction endonuclease cleavage sites of thepolynucleotides; determining mRNA and DNA localization of Zven1 or Zven2polynucleotides in tissues (i.e., by northern and Southern blotting aswell as polymerase chain reaction); and for identifying relatedpolynucleotides and polypeptides by nucleic acid hybridization. As anillustration, students will find that PvuII digestion of a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 389of SEQ ID NO:1 provides two fragments of about 123 base pairs, and 201base pairs, whereas HaeIII digestion yields fragments of about 46 basepairs, and 278 base pairs.

Zven1 or Zven2 polypeptides can be used as an aid to teach preparationof antibodies; identifying proteins by western blotting; proteinpurification; determining the weight of expressed Zven1 or Zven2polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., proteaseinhibition) in vitro and in vivo. For example, students will find thatdigestion of unglycosylated Zven1 with cyanogen bromide yields fourfragments having approximate molecular weights of 148, 4337, 1909, 2402,and 2939, whereas digestion of unglycosylated Zven1 with BNPS orNCS/urea yields fragments having approximate molecular weights of 5231,and 6444.

Zven1 or Zven2 polypeptides can also be used to teach analytical skillssuch as mass spectrometry, circular dichroism, to determineconformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing Zven1 or Zven2can be given to the student to analyze. Since the amino acid sequencewould be known by the instructor, the protein can be given to thestudent as a test to determine the skills or develop the skills of thestudent, the instructor would then know whether or not the student hascorrectly analyzed the polypeptide. Since every polypeptide is unique,the educational utility of Zven1 or Zven2 would be unique unto itself.

The antibodies which bind specifically to Zven1 or Zven2 can be used asa teaching aid to instruct students how to prepare affinitychromatography columns to purify Zven1 or Zven2, cloning and sequencingthe polynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. The Zven1 orZven2 gene, polypeptide, or antibody would then be packaged by reagentcompanies and sold to educational institutions so that the students gainskill in art of molecular biology. Because each gene and protein isunique, each gene and protein creates unique challenges and learningexperiences for students in a lab practicum. Such educational kitscontaining the Zven1 or Zven2 gene, polypeptide, or antibody areconsidered within the scope of the present invention.

For any Zven polypeptide, including variants and fusion proteins, one ofordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zven1 or Zven2 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6. Suitable forms of computer-readable media include magneticmedia and optically-readable media. Examples of magnetic media include ahard or fixed drive, a random access memory (RAM) chip, a floppy disk,digital linear tape (DLT), a disk cache, and a ZIP disk. Opticallyreadable media are exemplified by compact discs (e.g., CD-read onlymemory (ROM), CD-rewritable (RW), and CD-recordable), and digitalversatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

5. Production of Zven Fusion Proteins

Fusion proteins of Zven can be used to express a Zven polypeptide orpeptide in a recombinant host, and to isolate expressed Zvenpolypeptides and peptides. One type of fusion protein comprises apeptide that guides a Zven polypeptide from a recombinant host cell. Todirect a Zven polypeptide into the secretory pathway of a eukaryotichost cell, a secretory signal sequence (also known as a signal peptide,a leader sequence, prepro sequence or pre sequence) is provided in theZven expression vector. While the secretory signal sequence may bederived from Zven1 or Zven2, a suitable signal sequence may also bederived from another secreted protein or synthesized de novo. Thesecretory signal sequence is operably linked to a Zven1- orZven2-encoding sequence such that the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the nucleotide sequenceencoding the polypeptide of interest, although certain secretory signalsequences may be positioned elsewhere in the nucleotide sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of Zven1, Zven2, or anotherprotein produced by mammalian cells (e.g., tissue-type plasminogenactivator signal sequence, as described, for example, in U.S. Pat. No.5,641,655) is useful for expression of Zven1 or Zven2 in recombinantmammalian hosts, a yeast signal sequence is preferred for expression inyeast cells. Examples of suitable yeast signal sequences are thosederived from yeast mating phermone α-factor (encoded by the MFα1 gene),invertase (encoded by the SUC2 gene), or acid phosphatase (encoded bythe PHO5 gene). See, for example, Romanos et al., “Expression of ClonedGenes in Yeast,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zven1 orZven2 can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zven1 or Zven2fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

Another form of fusion protein comprises a Zven1 or Zven2 polypeptideand an immunoglobulin heavy chain constant region, typically an F_(C)fragment, which contains two constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:7). In this fusion protein, a preferred Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zven fusion protein that comprises a Zven1 or Zven2polypeptide moiety and a human Fc fragment, wherein the C-terminus ofthe Zven polypeptide moiety is attached to the N-terminus of the Fcfragment via a peptide linker, such as a peptide consisting of the aminoacid sequence of SEQ ID NO:7.

In another variation, a Zven1 or Zven2 fusion protein comprises an IgGsequence, a Zven polypeptide moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that iscovalently joined to the amino terminal of the Zven polypeptide moiety,wherein the IgG sequence consists of the following elements in thefollowing order: a hinge region, a CH₂ domain, and a CH₃ domain.Accordingly, the IgG sequence lacks a CH₁ domain. The Zven polypeptidemoiety displays a Zven1 or Zven2 activity, such as the ability to bindwith a Zven1 or Zven2 receptor. This general approach to producingfusion proteins that comprise both antibody and nonantibody portions hasbeen described by LaRochelle et al., EP 742830 (WO 95/21258).

Fusion proteins comprising a Zven1 or Zven2 polypeptide moiety and an Fcmoiety can be used, for example, as an in vitro assay tool. For example,the presence of a Zven1 or Zven2 receptor in a biological sample can bedetected using these Zven1 or Zven2-antibody fusion proteins, in whichthe Zven moiety is used to target the cognate receptor, and amacromolecule, such as Protein A or anti-Fc antibody, is used to detectthe bound fusion protein-ligand complex. In addition, antibody-Zvenfusion proteins, comprising antibody variable domains, are useful astherapeutic proteins, in which the antibody moiety binds with a targetantigen, such as a tumor associated antigen.

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

6. Production of Zven Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zven1 or Zven2 gene, a nucleic acid molecule encoding the polypeptidemust be operably linked to regulatory sequences that controltranscriptional expression in an expression vector and then, introducedinto a host cell. In addition to transcriptional regulatory sequences,such as promoters and enhancers, expression vectors can includetranslational regulatory sequences and a marker gene, which is suitablefor selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zven1 expression vector maycomprise a Zven1 gene and a secretory sequence derived from a Zven1 geneor another secreted gene.

Zven1 or Zven2 proteins of the present invention may be expressed inmammalian cells. Examples of suitable mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61;CHO DG44 [Chasin et al., Som. Cell. Molec. Genet. 12:555 1986]), ratpituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zven1 or Zven2 geneexpression in mammalian cells if the prokaryotic promoter is regulatedby a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990),and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zven1 or Zven2 polypeptides can also be produced by cultured mammaliancells using a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Zven1 or Zven2 genes may also be expressed in other higher eukaryoticcells, such as avian, fungal, insect, yeast, or plant cells. Thebaculovirus system provides an efficient means to introduce cloned Zven1or Zven2 genes into insect cells. Suitable expression vectors are basedupon the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV), and contain well-known promoters such as Drosophila heat shockprotein (hsp) 70 promoter, Autographa californica nuclear polyhedrosisvirus immediate-early gene promoter (ie-1) and the delayed early 39Kpromoter, baculovirus p10 promoter, and the Drosophila metallothioneinpromoter. A second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zven polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zven polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zven1 or Zven2 gene is transformed into E. coli, andscreened for bacmids, which contain an interrupted lacZ gene indicativeof recombinant baculovirus. The bacmid DNA containing the recombinantbaculovirus genome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native Zven1/Zven2 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native Zven1/Zven2 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which can be linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is possible to use host cells in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be used that are deficient in vacuolarprotease genes (PEP4 and PRB1). Electroporation is used to facilitatethe introduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zven genes can be expressed in prokaryotic host cells.Suitable promoters that can be used to express Zven1 or Zven2polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM10, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, M119, M1120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zven polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NOs:2 and 5. Illustrative polypeptides of Zven2, for example, include15 contiguous amino acid residues of amino acids 82 to 105 of SEQ IDNO:5. Exemplary polypeptides of Zven1 include 15 contiguous amino acidresidues of amino acids 1 to 32 or amino acids 75 to 108 of SEQ ID NO:2,whereas exemplary Zven2 polypeptides include amino acids 82 to 105 ofSEQ ID NO:5. Within certain embodiments of the invention, thepolypeptides comprise 20, 30, 40, 50, 75, or more contiguous residues ofSEQ ID NOs:2 or 5. Nucleic acid molecules encoding such peptides andpolypeptides are useful as polymerase chain reaction primers and probes.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

7. Isolation of Zven Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or even greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention can also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, a purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zven1 or Zven2 purified from natural sources, andrecombinant Zven polypeptides and fusion Zven polypeptides purified fromrecombinant host cells. In general, ammonium sulfate precipitation andacid or chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zven isolation and purification can be devisedby those of skill in the art. For example, anti-Zven antibodies,obtained as described below, can be used to isolate large quantities ofprotein by immunoaffinity purification. Moreover, methods for bindingreceptors to ligand polypeptides, such as Zven1 or Zven2, bound tosupport media are well known in the art.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Zven polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zven polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

8. Zven Analogs and Zven Receptors

As described above, the disclosed polypeptides can be used to constructZven variants. These polypeptides can be used to identify Zven1 or Zven2analogs. One type of Zven analog mimics Zven by binding with a Zvenreceptor. Such an analog is considered to be a Zven agonist if thebinding of the analog with a Zven receptor stimulates a response by acell that expresses the receptor. On the other hand, a Zven analog thatbinds with a Zven receptor, but does not stimulate a cellular response,may be a Zven antagonist. Such an antagonist may diminish Zven or Zvenagonist activity, for example, by a competitive or non-competitivebinding of the antagonist to the Zven receptor.

One general class of Zven analogs are agonists or antagonists having anamino acid sequence that is a mutation of the amino acid sequencesdisclosed herein. Another general class of Zven analogs is provided byanti-idiotype antibodies, and fragments thereof, as described below.Moreover, recombinant antibodies comprising anti-idiotype variabledomains can be used as analogs (see, for example, Monfardini et al.,Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domainsof anti-idiotype Zven antibodies mimic Zven, these domains can provideeither Zven agonist or antagonist activity. As an illustration, Lim andLanger, J. Interferon Res. 13:295 (1993), describe anti-idiotypicinterferon-α antibodies that have the properties of either interferon-αagonists or antagonists.

A third approach to identifying Zven1 or Zven2 analogs is provided bythe use of combinatorial libraries. Methods for constructing andscreening phage display and other combinatorial libraries are provided,for example, by Kay et al., Phage Display of Peptides and Proteins(Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al.,U.S. Pat. No. 5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

Zven1, Zven2, their agonists and antagonists are valuable in both invivo and in vitro uses. For example, Zven1, Zven2, or an agonist can beused as a component of defined cell culture media, alone or incombination with other bioactive agents, to replace serum that iscommonly used in cell culture. For example, Zven proteins can be used tomaintain in vitro models of spermatogenesis. Zven proteins can also beused to promote organ or tissue regeneration, to eliminate or to controlcell proliferation, or to fabricate matrix elements within a vascularprosthesis, for example, to promote remodeling of vessels from anartificial vessel implant.

Antagonists are also useful as research reagents for characterizingsites of interaction between a Zven polypeptide and its receptor. In atherapeutic setting, pharmaceutical compositions comprising Zvenantagonists can be used to inhibit Zven activity. As an illustration,Zven antagonists can be used to inhibit contraction of the ileum, and todecrease hyperalgesia.

The activity of a Zven polypeptide, agonist, or antagonist can bedetermined using a standard cell proliferation or differentiation assay.For example, assays measuring proliferation include such assays aschemosensitivity to neutral red dye, incorporation of radiolabelednucleotides, incorporation of 5-bromo-2′-deoxyuridine in the DNA ofproliferating cells, and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55 (1983); Porstmann et al., J. Immunol. Methods 82:169(1985); Alley et al., Cancer Res. 48:589 (1988); Cook et al., AnalyticalBiochem. 179:1 (1989); Marshall et al., Growth Reg. 5:69 (1995);Scudiero et al., Cancer Res. 48:4827 (1988); Cavanaugh et al.,Investigational New Drugs 8:347 (1990)). Assays measuringdifferentiation include, for example, measuring cell-surface markersassociated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Raes, Adv. Anim.Cell Biol. Technol. Bioprocesses, pages 161-171 (1989; Watt, FASEB,5:281 (1991); Francis, Differentiation 57:63 (1994)). Assays can be usedto measure other cellular responses, that include, chemotaxis, adhesion,changes in ion channel influx, regulation of second messenger levels andneurotransmitter release. Such assays are well known in the art (see,for example, Chayen and Bitensky, Cytochemical Bioassays: Techniques &Applications (Marcel Dekker 1983)).

The effect of a variant Zven polypeptide can also be determined byobserving contractility of tissues, including gastrointestinal tissues,with tensiometer that measures contractility and relaxation in tissues(see, for example, Dainty et al., J. Pharmacol. 100:767 (1990); Rhee etal., Neurotox. 16:179 (1995); Anderson, Endocrinol. 114:364 (1984);Downing, and Sherwood, Endocrinol. 116:1206 (1985)). For example,methods for measuring vasodilatation of aortic rings are well known inthe art. As an illustration, aortic rings are removed from four-monthold Sprague Dawley rats and placed in a buffer solution, such asmodified Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO₄.7H₂O,1.2 mM KH₂PO₄, 2.5 mM CaCl₂.2H₂O, 24.8 mM NaHCO₃ and 10 mM glucose). Oneof skill in the art would recognize that this method can be used withother animals, such as rabbits, other rat strains, Guinea pigs, and thelike. The rings are then attached to an isometric force transducer(Radnoti Inc., Monrovia, Calif.) and the data are recorded with aPonemah physiology platform (Gould Instrument systems, Inc., ValleyView, Ohio) and placed in an oxygenated (95% O₂, 5% CO₂) tissue bathcontaining the buffer solution. The tissues are adjusted to one gramresting tension and allowed to stabilize for about one hour beforetesting. The integrity of the rings can be tested with norepinepherin(Sigma Co.; St. Louis, Mo.) and carbachol, a muscarinic acetylcholineagonist (Sigma Co.). After integrity is checked, the rings are washedthree times with fresh buffer and allowed to rest for about one hour. Totest a sample for vasodilatation, or relaxation of the aortic ringtissue, the rings are contracted to two grams tension and allowed tostabilize for fifteen minutes. A Zven polypeptide sample is then addedto one, two, or three of the four baths, without flushing, and tensionon the rings recorded and compared to the control rings containingbuffer only. Enhancement or relaxation of contractility by Zvenpolypeptides, their agonists and antagonists is directly measured bythis method, and it can be applied to other contractile tissues such asgastrointestinal tissues.

The effect of a variant Zven polypeptide on gastric motility wouldtypically be measured in the clinical setting as the time required forgastric emptying and subsequent transit time through thegastrointestinal tract. Gastric emptying scans are well known to thoseskilled in the art, and briefly, comprise use of an oral contrast agent,such as barium, or a radiolabeled meal. Solids and liquids can bemeasured independently. Generally, a test food or liquid is radiolabeledwith an isotope (e.g., 99 mTc), and after ingestion or administration,transit time through the gastrointestinal tract and gastric emptying aremeasured by visualization using gamma cameras (Meyer et al., Am. J. Dig.Dis. 21:296 (1976); Collins et al., Gut 24:1117 (1983); Maughan et al.,Diabet. Med. 13:S6 (1996), and Horowitz et al., Arch. Intern. Med.145:1467 (1985)). These studies can be performed before and after theadministration of a promotility agent to quantify the efficacy of theZven polypeptide.

To determine if a variant Zven polypeptide is a chemotractant in vivo,the Zven polypeptide can be administered by intradermal orintraperitoneal injection. Characterization of the accumulatedleukocytes at the site of injection can be determined using lineagespecific cell surface markers and fluorescence immunocytometry or byimmunohistochemistry (see, for example, Jose, J. Exp. Med. 179:881(1994)). Release of specific leukocyte cell populations from bone marrowinto peripheral blood can also be measured after Zven injection.

Zven1 or Zven2 polypeptides can be used to identify and to isolate theircognate receptors. For example, proteins and peptides of the presentinvention can be immobilized on a column and used to bind receptors froma biological sample that is run over the column (Hermanson et al.(eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (AcademicPress 1992)). As a receptor ligand, the activity of Zven1 or Zven2 canbe measured by a silicon-based biosensor microphysiometer, whichmeasures the extracellular acidification rate or proton excretionassociated with receptor binding and subsequent cellular responses. Anexemplary device is the CYTOSENSOR Microphysiometer manufactured byMolecular Devices Corp. (Sunnyvale, Calif.). A variety of cellularresponses, such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method (see, for example, McConnell et al.,Science 257:1906 (1992), Pitchford et al., Meth. Enzymol. 228:84 (1997),Arimilli et al., J. Immunol. Meth. 212:49 (1998), and Van Liefde et al.,Eur. J. Pharmacol. 346:87 (1998)). Moreover, the microphysiometer can beused for assaying adherent or non-adherent eukaryotic or prokaryoticcells.

Since energy metabolism is coupled with the use of cellular ATP, anyevent, which alters cellular ATP levels, such as receptor activation andthe initiation of signal transduction, will cause a change in cellularacid section. By measuring extracellular acidification changes in cellmedia over time, therefore, the microphysiometer directly measurescellular responses to various stimuli, including Zven1, Zven2, theiragonists, or antagonists. The microphysiometer can be used to measureresponses of a Zven-responsive eukaryotic cell, compared to a controleukaryotic cell that does not respond to a Zven polypeptide.Zven-responsive eukaryotic cells comprise cells into which a Zvenreceptor has been transfected to create a cell that is responsive toZven, or cells that are naturally responsive to Zven. Zven-modulatedcellular responses are measured by a change (e.g., an increase ordecrease in extracellular acidification) in the response of cellsexposed to Zven1 or Zven2, compared with control cells that have notbeen exposed to Zven1 or Zven2.

Accordingly, a microphysiometer can be used to identify cells, tissues,or cell lines which respond to a Zven-stimulated pathway, and whichexpress a functional Zven receptor. As an illustration, cells thatexpress a functional Zven1 receptor can be identified by (a) providingtest cells, (b) incubating a first portion of the test cells in theabsence of Zven1, (c) incubating a second portion of the test cells inthe presence of Zven1, and (d) detecting a change (e.g., an increase ordecrease in extracellular acidification rate, as measured by amicrophysiometer) in a cellular response of the second portion of thetest cells, as compared to the first portion of the test cells, whereinsuch a change in cellular response indicates that the test cells expressa functional Zven1 receptor. An additional negative control may beincluded in which a portion of the test cells is incubated with Zven1and an anti-Zven1 antibody to inhibit the binding of Zven1 with itscognate receptor. Similar approaches can be used to identify cells thatexpress a functional Zven2 receptor

Radiolabeled or affinity labeled Zven polypeptides can also be used toidentify or to localize Zven receptors in a biological sample (see, forexample, Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37(Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62:483 (1993);Fedan et al., Biochem. Pharmacol. 33:1167 (1984)). Also see, Varthakaviand Minocha, J. Gen. Virol. 77:1875 (1996), who describe the use ofanti-idiotype antibodies for receptor identification.

A Zven polypeptide or Zven fusion protein can be immobilized onto thesurface of a receptor chip of a commercially available biosensorinstrument (BIACORE, Biacore AB; Uppsala, Sweden). The use of thisinstrument is disclosed, for example, by Karlsson, Immunol. Methods145:229 (1991), and Cunningham and Wells, J. Mol. Biol. 234:554 (1993).This approach can be used to identify a Zven receptor, or an agonist orantagonist of a Zven receptor.

Zven1 or Zven2 receptor binding domains can be further characterized byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of Zven1 or Zven2 agonists. See, for example, de Vos etal., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992),and Wlodaver et al., FEBS Lett. 309:59 (1992).

9. Production of Antibodies to Zven Proteins

Antibodies to a Zven polypeptide can be obtained, for example, using theproduct of a Zven expression vector or Zven isolated from a naturalsource as an antigen. Particularly useful anti-Zven1 and anti-Zven2antibodies “bind specifically” with Zven1 and Zven2, respectively.Antibodies are considered to be specifically binding if the antibodiesexhibit at least one of the following two properties: (1) antibodiesbind to Zven1 or Zven2 with a threshold level of binding activity, and(2) antibodies do not significantly cross-react with polypeptidesrelated to Zven1 or Zven2.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zven polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zven, but not knownpolypeptides (e.g., known Wnt inhibitors) using a standard Western blotanalysis. Particular anti-Zven1 antibodies bind Zven1, but not Zven2,while certain anti-Zven2 antibodies bind Zven2, but not Zven1.

Anti-Zven1 and anti-Zven2 antibodies can be produced using antigenicZven1 or Zven2 epitope-bearing peptides and polypeptides. Antigenicepitope-bearing peptides and polypeptides of the present inventioncontain a sequence of at least four, or between 15 to about 30 aminoacids contained within SEQ ID NOs:2 or 5. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence ofthe invention, containing from 30 to 50 amino acids, or any length up toand including the entire amino acid sequence of a polypeptide of theinvention, also are useful for inducing antibodies that bind with Zven1or Zven2. It is desirable that the amino acid sequence of theepitope-bearing peptide is selected to provide substantial solubility inaqueous solvents (i.e., the sequence includes relatively hydrophilicresidues, while hydrophobic residues are preferably avoided). Moreover,amino acid sequences containing proline residues may be also bedesirable for antibody production.

As an illustration, potential antigenic sites in Zven1 or Zven2 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that suitable antigenic peptidesof Zven1 include the following segments of the amino acid sequence ofSEQ ID NO:2: amino acids 22 to 27 (“antigenic peptide 1”), amino acids33 to 41 (“antigenic peptide 2”), amino acids 61 to 68 (“antigenicpeptide 3”), amino acids 80 to 85 (“antigenic peptide 4”), amino acids97 to 102 (“antigenic peptide 5”), and amino acids 61 to 85 (“antigenicpeptide 6”). The present invention contemplates the use of any one ofantigenic peptides 1 to 6 to generate antibodies to Zven1. The presentinvention also contemplates polypeptides comprising at least one ofantigenic peptides 1 to 6.

Similarly, analysis of the Zven2 amino acid sequence indicated thatsuitable antigenic peptides of Zven2 include the following segments ofthe amino acid sequence of SEQ ID NO:5: amino acids 25 to 33 (“antigenicpeptide 7”), amino acids 53 to 66 (“antigenic peptide 8”), amino acids88 to 95 (“antigenic peptide 9”), amino acids 98 to 103 (“antigenicpeptide 10”), and amino acids 88 to 103 (“antigenic peptide 11”). Thepresent invention contemplates the use of any one of antigenic peptides7 to 11 to generate antibodies to Zven2. The present invention alsocontemplates polypeptides comprising at least one of antigenic peptides7 to 11.

Polyclonal antibodies to recombinant Zven protein or to Zven isolatedfrom natural sources can be prepared using methods well-known to thoseof skill in the art. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press 1992), and Williams et al., “Expression of foreignproteins in E. coli using plasmid vectors and purification of specificpolyclonal antibodies,” in DNA Cloning 2: Expression Systems, 2ndEdition, Glover et al. (eds.), page 15 (Oxford University Press 1995).The immunogenicity of a Zven polypeptide can be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of Zven or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zven antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zven antibodies can be generated. Rodentmono-clonal antibodies to specific antigens may be obtained by methodsknown to those skilled in the art (see, for example, Kohler et al.,Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zven gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zven antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zven antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains, which areconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991) (alsosee, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZven polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled Zven protein or peptide). Genes encoding polypeptides havingpotential Zven polypeptide binding domains can be obtained by screeningrandom peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides, which interactwith a known target which can be a protein or polypeptide, such as aligand or receptor, a biological or synthetic macromolecule, or organicor inorganic substances. Techniques for creating and screening suchrandom peptide display libraries are known in the art (Ladner et al.,U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladneret al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,and Kay et al., Phage Display of Peptides and Proteins (Academic Press,Inc. 1996)) and random peptide display libraries and kits for screeningsuch libraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zven sequences disclosed herein to identifyproteins which bind to Zven.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zven antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat'l Acad.Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992),Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun.150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zven antibodies or antibody fragments, using standardtechniques. See, for example, Green et al., “Production of PolyclonalAntisera,” in Methods In Molecular Biology: Immunochemical Protocols,Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can beprepared using anti-Zven antibodies or antibody fragments as immunogenswith the techniques, described above. As another alternative, humanizedanti-idiotype antibodies or subhuman primate anti-idiotype antibodiescan be prepared using the above-described techniques. Methods forproducing anti-idiotype antibodies are described, for example, by Irie,U.S. Pat. No. 5,208,146, Greene, et. al., U.S. Pat. No. 5,637,677, andVarthakavi and Minocha, J. Gen. Virol. 77:1875 (1996).

10. Detection of Zven Gene Expression and Examination of the ZvenChromosomal Locus

Nucleic acid molecules can be used to detect the expression of a Zven1or Zven2 gene in a biological sample. Such probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NOs:1 or 4, or a fragment thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NOs:1 or 4, or a fragment thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like.

Illustrative probes comprise a portion of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 334 to 405 of SEQ ID NO:4, or to the complement of suchnucleotide sequences. An additional example of a suitable probe is aprobe consisting of nucleotides 354 to 382 of SEQ ID NO:1, or a portionthereof. As used herein, the term “portion” refers to at least eightnucleotides to at least 20 or more nucleotides.

For example, nucleic acid molecules comprising a portion of thenucleotide sequence of SEQ ID NO:1 can be used to detect activatedneutrophils. Such molecules can also be used to identity therapeutic orprophylactic agents that modulate the response of a neutrophil to apathogen.

In a detection basic assay, a single-stranded probe molecule isincubated with RNA, isolated from a biological sample, under conditionsof temperature and ionic strength that promote base pairing between theprobe and target Zven RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zven RNAcan be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zven oligonucleotide probes are also useful for in vivo diagnosis. As anillustration, ¹⁸F-labeled oligonucleotides can be administered to asubject and visualized by positron emission tomography (Tavitian et al.,Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated with Zvenprimers (see, for example, Wu et al. (eds.), “Rapid Isolation ofSpecific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages15-28 (CRC Press, Inc. 1997)). PCR is then performed and the productsare analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the guanidinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zven anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zven sequences are amplified by thepolymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zven probe, and examinedby autoradiography. Additional alternative approaches include the use ofdigoxigenin-labeled deoxyribonucleic acid triphosphates to providechemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach for detection of Zven1 or Zven2 expression is cyclingprobe technology (CPT), in which a single-stranded DNA target binds withan excess of DNA-RNA-DNA chimeric probe to form a complex, the RNAportion is cleaved with RNAase H, and the presence of cleaved chimericprobe is detected (see, for example, Beggs et al., J. Clin. Microbiol.34:2985 (1996), Bekkaoui et al., Biotechniq es 20:240 (1996)).Alternative methods for detection of Zven sequences can utilizeapproaches such as nucleic acid sequence-based amplification (NASBA),cooperative amplification of templates by cross-hybridization (CATCH),and the ligase chain reaction (LCR) (see, for example, Marshall et al.,U.S. Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161(1996), Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick etal., J. Virol. Methods 70:59 (1998)). Other standard methods are knownto those of skill in the art.

Zven probes and primers can also be used to detect and to localize Zvengene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)).

The Zven2 gene was found to reside at human chromosome 1p13; the Wnt2Bgene also resides in this region, as well as differentiation genes CSF1and Notch2. Chromosome 1p13 is associated with various diseases anddisorders, including retinitis pigmentosa, Stargardt disease,Waardenburg syndrome, nemaline myopathy, Kabuki syndrome, andcardiomyopathy. The Zven1 gene resides in human chromosome3p21.1-3p14.3. This region of chromosome 3 is associated withmetaphyseal chondrodysplasia, small cell cancer of the lung, cerebralgigantism (Sotos Syndrome), Larsen Syndrome, spinocerebellar ataxia,Wemicke-Korsakoff Syndrome, hyperglycinemia, septooptic dysplasia,progressive external ophthalmoplegia, and pancreatic cancer. The Wnt5Agene also resides in this region.

Nucleic acid molecules comprising Zven nucleotide sequences can be usedto determine whether a subject's chromosomes contain a mutation in theZven gene. Detectable chromosomal aberrations at the Zven1 or Zven2 genelocus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Of particular interest are genetic alterations thatinactivate a Zven1 or Zven2 gene.

Aberrations associated with a Zven1 or Zven2 locus can be detected usingnucleic acid molecules of the present invention by employing moleculargenetic techniques, such as restriction fragment length polymorphismanalysis, short tandem repeat analysis employing PCR techniques,amplification-refractory mutation system analysis, single-strandconformation polymorphism detection, RNase cleavage methods, denaturinggradient gel electrophoresis, fluorescence-assisted mismatch analysis,and other genetic analysis techniques known in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis,Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) MolecularDiagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren(ed.), Laboratory Protocols for Mutation Detection (Oxford UniversityPress 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: DetectingGenes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998),and Richards and Ward, “Molecular Diagnostic Testing,” in Principles ofMolecular Medicine, pages 83-88 (Humana Press, Inc. 1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zven target sequenceand to introduce an RNA polymerase promoter, a translation initiationsequence, and an in-frame ATG triplet. PCR products are transcribedusing an RNA polymerase, and the transcripts are translated in vitrowith a T7-coupled reticulocyte lysate system. The translation productsare then fractionated by SDS-PAGE to determine the lengths of thetranslation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

The present invention also contemplates kits for performing a diagnosticassay for Zven1 or Zven2 gene expression or to examine a Zven locus.Such kits comprise nucleic acid probes, such as double-stranded nucleicacid molecules comprising the nucleotide sequence of SEQ ID NOs:1 or 4,or a fragment thereof, as well as single-stranded nucleic acid moleculeshaving the complement of the nucleotide sequence of SEQ ID NOs:1 or 4,or a fragment thereof. Probe molecules may be DNA, RNA,oligonucleotides, and the like. Kits may comprise nucleic acid primersfor performing PCR.

Such a kit can contain all the necessary elements to perform a nucleicacid diagnostic assay described above. A kit will comprise at least onecontainer comprising a Zven probe or primer. The kit may also comprise asecond container comprising one or more reagents capable of indicatingthe presence of Zven sequences. Examples of such indicator reagentsinclude detectable labels such as radioactive labels, fluorochromes,chemiluminescent agents, and the like. A kit may also comprise a meansfor conveying to the user that the Zven probes and primers are used todetect Zven gene expression. For example, written instructions may statethat the enclosed nucleic acid molecules can be used to detect either anucleic acid molecule that encodes Zven, or a nucleic acid moleculehaving a nucleotide sequence that is complementary to a Zven-encodingnucleotide sequence. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

11. Detection of Zven Protein with Anti-Zven Antibodies

The present invention contemplates the use of anti-Zven antibodies toscreen biological samples in vitro for the presence of Zven1 or Zven2.In one type of in vitro assay, anti-Zven antibodies are used in liquidphase. For example, the presence of Zven in a biological sample can betested by mixing the biological sample with a trace amount of labeledZven1 (or Zven2) and an anti-Zven antibody under conditions that promotebinding between Zven and its antibody. Complexes of Zven and anti-Zvenin the sample can be separated from the reaction mixture by contactingthe complex with an immobilized protein which binds with the antibody,such as an Fc antibody or Staphylococcus protein A. The concentration ofZven in the biological sample will be inversely proportional to theamount of labeled Zven bound to the antibody and directly related to theamount of free labeled Zven.

Alternatively, in vitro assays can be performed in which anti-Zvenantibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zven antibodies can be used to detect Zven1 orZven2 in tissue sections prepared from a biopsy specimen. Suchimmunochemical detection can be used to determine the relative abundanceof Zven and to determine the distribution of Zven in the examinedtissue. General immunochemistry techniques are well established (see,for example, Ponder, “Cell Marking Techniques and Their Application,” inMammalian Development: A Practical Approach, Monk (ed.), pages 115-38(IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), MethodsIn Molecular Biology, Vol. 10: Immunochemical Protocols (The HumanaPress, Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zven antibody, and then contacting the biologicalsample with a detectably labeled molecule which binds to the antibody.For example, the detectably labeled molecule can comprise an antibodymoiety that binds to anti-Zven antibody. Alternatively, the anti-Zvenantibody can be conjugated with avidin/streptavidin (or biotin) and thedetectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zven antibody can be conjugated with a detectablelabel to form an anti-Zven immunoconjugate. Suitable detectable labelsinclude, for example, a radioisotope, a fluorescent label, achemiluminescent label, an enzyme label, a bioluminescent label orcolloidal gold. Methods of making and detecting such detectably-labeledimmunoconjugates are well-known to those of ordinary skill in the art,and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zven immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zven immunoconjugates can be detectably labeled bycoupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zvenimmunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zven immunoconjugates can be detectably labeled bylinking an anti-Zven antibody component to an enzyme. When theanti-Zven-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels, which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zven antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al, Clin. Chim. Acta 70:1 (1976), Schurs etal, Clin. Chim. Acta 81:1 (1977), Shih et al, Int'l Cancer46:1101(1990),Stein et al., Cancer Res. 50:1330(1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zven antibodies that have been conjugatedwith avidin, streptavidin, and biotin (see, for example, Wilchek et al.(eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184(Academic Press 1990), and Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10,Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

In a related approach, biotin- or FITC-labeled Zven1 or Zven2 can beused to identify cells that bind Zven1 or Zven2. Such can binding can bedetected, for example, using flow cytometry.

The present invention also contemplates kits for performing animmunological diagnostic assay for Zven gene expression. Such kitscomprise at least one container comprising an anti-Zven antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zven antibodyor antibody fragments. Examples of such indicator reagents includedetectable labels such as a radioactive label, a fluorescent label, achemiluminescent label, an enzyme label, a bioluminescent label,colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zven antibodies or antibody fragments areused to detect Zven protein. For example, written instructions may statethat the enclosed antibody or antibody fragment can be used to detectZven. The written material can be applied directly to a container, orthe written material can be provided in the form of a packaging insert.

12. Therapeutic Uses of Zven Polypeptides

The present invention includes the use of proteins, polypeptides, andpeptides having Zven activity (such as Zven polypeptides, Zven analogs,active Zven anti-idiotype antibodies, and Zven fusion proteins) to asubject, which lacks an adequate amount of this polypeptide. The presentinvention contemplates both veterinary and human therapeutic uses.Illustrative subjects include mammalian subjects, such as farm animals,domestic animals, and human patients.

For example, a protein, a polypeptide, or a peptide having Zven1activity can be administered to a subject (e.g., a human patient), whichhas small cell cancer of the lung. In contrast, Zven antagonists (e.g.,anti-Zven antibodies or anti-Zven anti-idiotype antibodies that arebiologically inactive) can be used to treat a subject who produces anexcess of Zven. Therapeutic uses for Zven proteins include, anti-tumoragent (e.g., anti-lung tumor agent), anti-inflammatory agent, an agentto regulate regeneration or remodeling of tissues, and an agent tomodulate necrosis or tissue growth developmental arrest. As anillustration, Zven polypeptides may be used to promote wound healing, toprevent or to treat an adverse reaction of the skin to askin-sensitizing agent or a skin-irritating agent, or to stimulate theimmune system of an immunocompromised individual.

For example, polypeptides, peptides, and peptides having Zven2 activitymay be used to inhibit cellular proliferation, cellular differentiation,and necrosis. In particular, polypeptides, peptides, and peptides havingZven2 activity may be used to inhibit cellular proliferation associatedwith mammary tumors, colon cancer, melanomas, hepatocellular carcinomas,and the like.

The Zven polypeptides of the present invention may also be used intreatment of disorders associated with gastrointestinal cellcontractility, secretion of digestive enzymes and acids,gastrointestinal motility, recruitment of digestive enzymes;inflammation, particularly as it affects the gastrointestinal system;and reflux disease and regulation of nutrient absorption; and modulationof blood pressure. Specific conditions that will benefit from treatmentwith molecules of the present invention include, but are not limited to,diabetic gastroparesis, post-surgical gastroparesis, vagotomy, chronicidiopathic intestinal pseudo-obstruction and gastroesophageal refluxdisease. Additional uses include, gastric emptying for radiologicalstudies, stimulating gallbladder contraction and antrectomy. Zvenantagonists are useful for clinical conditions associated withgastrointestinal hypermotility such as diarrhea and Crohn's disease.

Generally, the dosage of administered polypeptide, protein or peptidewill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history.Typically, it is desirable to provide the recipient with a dosage of amolecule having Zven activity, which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of a molecule having Zven activity to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses. Alternatively,Zven1 or Zven2 can be administered as a controlled release formulation.

Additional routes of administration include oral, dermal,mucosal-membrane, pulmonary, and transcutaneous. Oral delivery issuitable for polyester microspheres, zein microspheres, proteinoidmicrospheres, polycyanoacrylate microspheres, and lipid-based systems(see, for example, DiBase and Morrel, “Oral Delivery ofMicroencapsulated Proteins,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). Thefeasibility of an intranasal delivery is exemplified by such a mode ofinsulin administration (see, for example, Hinchcliffe and Illum, Adv.Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingZven1 or Zven2 can be prepared and inhaled with the aid of dry-powderdispersers, liquid aerosol generators, or nebulizers (e.g., Pettit andGombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev.35:235 (1999)). This approach is illustrated by the AERX diabetesmanagement system, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer Zven1 or Zven2(Potts et al., Pharm. Biotechnol. 10:213 (1997)).

Zven proteins can also be applied topically as, for example, liposomalpreparations, gels, salves, as a component of a glue, prosthesis, orbandage, and the like. Topical administration is useful for woundhealing applications, including the prevention of excess scaring andgranulation tissue, prevention of keyloids, and prevention of adhesionsfollowing surgery.

A pharmaceutical composition comprising molecules having Zven1 or Zven2activity can be furnished in liquid form, in an aerosol, or in solidform. Proteins having Zven1 or Zven2 activity can be administered as aconjugate with a pharmaceutically acceptable water-soluble polymermoiety. As an illustration, a Zven1-polyethylene glycol conjugate isuseful to increase the circulating half-life of the interferon, and toreduce the immunogenicity of the polypeptide. Liquid forms, includingliposome-encapsulated formulations, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms, such as a miniosmotic pump or animplant. Other dosage forms can be devised by those skilled in the art,as shown, for example, by Ansel and Popovich, Pharmaceutical DosageForms and Drug Delivery Systems, 5^(th) Edition (Lea & Febiger 1990),Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^(th) Edition(Mack Publishing Company 1995), and by Ranade and Hollinger, DrugDelivery Systems (CRC Press 1996).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zven1 or Zven2 activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having Zven1 or Zven2 activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zven activity and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient.

For example, the present invention includes methods of inhibiting theproliferation of tumor cells, comprising the step of administering acomposition comprising a Zven2 polypeptide or peptide to the tumorcells. In an in vivo approach, the composition is a pharmaceuticalcomposition, administered in a therapeutically effective amount to amammalian subject, which has a tumor. Such in vivo administration canprovide at least one physiological effect selected from the groupconsisting of decreased number of tumor cells, decreased metastasis,decreased size of a solid tumor, and increased necrosis of a tumor.

A pharmaceutical composition comprising molecules having Zven activitycan be furnished in liquid form, or in solid form. Liquid forms,including liposome-encapsulated formulations, are illustrated byinjectable solutions and oral suspensions. Exemplary solid forms includecapsules, tablets, and controlled-release forms, such as a miniosmoticpump or an implant. Other dosage forms can be devised by those skilledin the art, as shown, for example, by Ansel and Popovich, PharmaceuticalDosage Forms and Drug Delivery Systems, 5^(th) Edition (Lea & Febiger1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^(th)Edition (Mack Publishing Company 1995), and by Ranade and Hollinger,Drug Delivery Systems (CRC Press 1996).

Zven1 or Zven2 pharmaceutical compositions may be supplied as a kitcomprising a container that comprises Zven1 or Zven2, a Zven1 or Zven2agonist, or a Zven1 or Zven2 antagonist (e.g., an anti-Zven1 or Zven2antibody or antibody fragment). For example, Zven1 or Zven2 can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Alternatively, such a kit can include a dry-powder disperser,liquid aerosol generator, or nebulizer for administration of atherapeutic polypeptide. Such a kit may further comprise writteninformation on indications and usage of the pharmaceutical composition.Moreover, such information may include a statement that the Zven1 orZven2 composition is contraindicated in patients with knownhypersensitivity to Zven1 or Zven2.

13. Therapeutic Uses of Zven Nucleotide Sequences

The present invention includes the use of Zven nucleotide sequences toprovide Zven amino acid sequences to a subject in need of proteins,polypeptides, or peptides having Zven activity, as discussed in theprevious section. For example, Zven2 nucleotide sequences can be used toproduce Zven2 in order to inhibit cellular proliferation. In addition, atherapeutic expression vector can be provided that inhibits Zven geneexpression, such as an anti-sense molecule, a ribozyme, or an externalguide sequence molecule.

There are numerous approaches to introduce a Zven gene to a subject,including the use of recombinant host cells that express Zven, deliveryof naked nucleic acid encoding Zven, use of a cationic lipid carrierwith a nucleic acid molecule that encodes Zven, and the use of virusesthat express Zven, such as recombinant retroviruses, recombinantadeno-associated viruses, recombinant adenoviruses, and recombinantHerpes simplex viruses [HSV] (see, for example, Mulligan, Science260:926 (1993), Rosenberg et al., Science 242:1575 (1988), LaSalle etal., Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivoapproach, for example, cells are isolated from a subject, transfectedwith a vector that expresses a Zven gene, and then transplanted into thesubject.

In order to effect expression of a Zven gene, an expression vector isconstructed in which a nucleotide sequence encoding a Zven gene isoperably linked to a core promoter, and optionally a regulatory element,to control gene transcription. The general requirements of an expressionvector are described above.

Alternatively, a Zven gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant HSV can be prepared in Verocells, as described by Brandt et al., J. Gen. Virol. 72:2043 (1991),Herold et al., J. Gen. Virol. 75:1211 (1994), Visalli and Brandt,Virology 185:419 (1991), Grau et al., Invest. Ophthalmol. Vis. Sci.30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992), and byBrown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a Zven gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration of Zvennucleic acid molecules. For example, Aihara and Miyazaki, NatureBiotechnology 16:867 (1998), have demonstrated the use of in vivoelectroporation for gene transfer into muscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zven anti-sense RNA that inhibits the expression of Zven.Suitable sequences for Zven anti-sense molecules can be derived from thenucleotide sequences of Zven disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zven mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode a Zvengene. According to this approach, an external guide sequence can beconstructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to ZvenmRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zven nucleotide acid sequence, such as a recombinant virus,will vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Suitable routes of administration of therapeutic vectors includeintravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zven gene in alltissues or under the control of a tissue-specific or tissue-preferredregulatory element. These over-producers of Zven can be used tocharacterize the phenotype that results from over-expression, and thetransgenic animals can serve as models for human disease caused byexcess Zven. Transgenic mice that over-express Zven also provide modelbioreactors for production of Zven in the milk or blood of largeranimals. Methods for producing transgenic mice are well-known to thoseof skill in the art (see, for example, Jacob, “Expression and Knockoutof Interferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), Monastersky and Robl (eds.), Strategies in TransgenicAnimal Science (ASM Press 1995), and Abbud and Nilson, “RecombinantProtein Expression in Transgenic Mice,” in Gene Expression Systems:Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.),pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZven gene can begin with adult, fertile males (studs) (B6C3f1, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zven encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zven1encoding sequences can comprise nucleotide sequences that encode aminoacid residues 23 to 108 of SEQ ID NO:2, while Zven2 encoding sequencescan encode a polypeptide comprising amino acid residues 148 to 405 ofSEQ ID NO:5.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zven gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zven mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express Zven, it isuseful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zven. As discussed above, Zven geneexpression can be inhibited using anti-sense genes, ribozyme genes, orexternal guide sequence genes. To produce transgenic mice thatunder-express the Zven gene, such inhibitory sequences are targeted toZven mRNA. Methods for producing transgenic mice that have abnormallylow expression of a particular gene are known to those in the art (see,for example, Wu et al., “Gene Underexpression in Cultured Cells andAnimals by Antisense DNA and RNA Strategies,” in Methods in GeneBiotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zven gene expression is to generate mice having at least one normalZven allele replaced by a nonfunctional Zven gene. One method ofdesigning a nonfunctional Zven gene is to insert another gene, such as aselectable marker gene, within a nucleic acid molecule that encodesZven. Standard methods for producing these so-called “knockout mice” areknown to those skilled in the art (see, for example, Jacob, “Expressionand Knockout of Interferons in Transgenic Mice,” in Overexpression andKnockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124(Academic Press, Ltd. 1994), and Wu et al., “New Strategies for GeneKnockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press1997)).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Expression of the Zven1 Gene

Zven1 gene expression was examined using a PCR array panel of celllines, including blood cell and connective tissue cell lines. In onestudy, Zven1 expression was found to be restricted to B cell, T cell,monocyte, and granulocyte cell lines. Zven1 appeared to be highlyexpressed in the promyelocytic cell line HL60. This observationindicates that Zven1 is expressed in blood progenitor cells, because theHL60 line is capable of differentiating into either monocytes orgranulocytes. The only tested nonhematopoietic line displaying Zven1expression was A549, a lung adenocarcinoma line.

In another study, freshly isolated human neutrophils and monocytes werescreened via PCR for Zven1 expression with or without lipopolysaccharide(LPS) activation. Zven1 gene expression was detected in unactivatedmonocytes, but not in activated monocytes. Expression was also apparentin granulocytes. Zven1 expression was not detected in endothelial cellsof a microvascular origin.

EXAMPLE 2 Inhibition of Cellular Proliferation by Zven1

The effect of Zven1 on cellular proliferation was examined usingconditioned media either from cells infected with an adenovirus vectordesigned to express Zven1, or from cells infected with an adenovirusvector that lacked a Zven1 gene (parental control). In one study, humanfibroblast cells from normal lung (ATCC NO. CRL-1490) were plated at2500 cells/100 □l/well in 96 well plates with normal growth medium (MEMwith Earle's salts and NEAA, 10% fetal bovine serum (FBS)). Afterplating, the cells were allowed to adhere to the plates for 24 hours.Media were then discarded, and conditioned media test samples diluted ingrowth media were added (100 □l/well). For comparison, murine Lewis Lungcarcinoma cells (8000 cells per well in 10 □l) were transferred into 96well plates, which contained 100 □l/well of conditioned media testsamples diluted in growth media (DMEM high glucose, 10% FBS). All cellswere incubated for 72 hours.

After 72 hours, cells were examined using the CellTiter 96®Non-Radioactive Cell Proliferation Assay (Promega Corporation; Madison,Wis.). Absorbance readings were measured at A572-A650. Percentinhibition values were calculated as the average of triplicate readingsof A572-A650, using the equation: 100−((100*Abs of sample)/Abs of mediumalone). The results indicated that Zven1 can inhibit the proliferationof Lewis Lung cells by about 50% below controls, whereas Zven1 treatmentappeared to inhibit the proliferation of normal lung cells by about 10%.

The ability of Zven1 to affect the proliferation of A549 human lungadenocarcinoma cells was tested with conditioned media. A549 cells areplated at 1,000 cells per well in Hams F12 containing 10% FBS, andincubated for three days prior to serum starvation in Hams F12 (withoutFBS) for 24 hours. Zven1 conditioned media samples were diluted 1:1 witheither serum-free media or media containing 10% FBS, and proliferationwas measured after a 72 hour incubation. The results of this studyindicated that Zven1 can inhibit the proliferation of A549 cells belowcontrols by about 25%.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polypeptide, comprising a sequence having at least 95%sequence identity with residues 23 to 108 of SEQ ID NO:2, wherein thepolypeptide stimulates gastrointestinal contractility.
 2. The isolatedpolypeptide of claim 1 comprising an affinity tag.
 3. The isolatedpolypeptide of claim 2, wherein the affinity tag is selected from thegroup consisting of: polyhistidine tract, protein A, glutathione Stransferase, Glu-Glu affinity tag, substance P, Flag peptide,streptavidin binding peptide, and maltose-binding protein.
 4. Theisolated polypeptide of claim 1, wherein the polypeptide comprises asequence having at least 96% sequence identity with residues 23 to 108of SEQ ID NO:
 2. 5. The isolated polypeptide of claim 1, wherein thepolypeptide comprises a sequence having at least 97% sequence identitywith residues 23 to 108 of SEQ ID NO:
 2. 6. The isolated polypeptide ofclaim 1, wherein the polypeptide comprises a sequence having at least98% sequence identity with residues 23 to 108 of SEQ ID NO:
 2. 7. Theisolated polypeptide of claim 1, wherein the polypeptide comprises asequence having at least 99% sequence identity with residues 23 to 108of SEQ ID NO:
 2. 8. The isolated polypeptide of claim 1, wherein thepolypeptide having at least 95% sequence identity with residues 23-108of SEQ ID NO: 2 comprises one, two, or three, or four conservative aminoacid substitutions, wherein the conservative amino acid substitution isa substitution among amino acids selected within each of the followinggroups: (a) glycine, alanine, valine, leucine, and isoleucine; (b)phenylalanine, tyrosine, and tryptophan; (c) serine and threonine; (d)aspartate and glutamate; (e) glutamine and asparagine; and (f) lysine,arginine and histidine.
 9. The isolated polypeptide of claim 4, whereinthe polypeptide having at least 96% sequence identity with residues23-108 of SEQ ID NO: 2 comprises one, two, or three conservative aminoacid substitutions, wherein the conservative amino acid substitution isa substitution among amino acids selected within each of the followinggroups: (a) glycine, alanine, valine, leucine, and isoleucine; (b)phenylalanine, tyrosine, and tryptophan; (c) serine and threonine; (d)aspartate and glutamate; (e) glutamine and asparagine; and (f) lysine,arginine and histidine.
 10. The isolated polypeptide of claim 5, whereinthe polypeptide having at least 97% sequence identity with residues23-108 of SEQ ID NO: 2 comprises one or two conservative amino acidsubstitutions, wherein the conservative amino acid substitution is asubstitution among amino acids selected within each of the followinggroups: (a) glycine, alanine, valine, leucine, and isoleucine; (b)phenylalanine, tyrosine, and tryptophan; (e) serine and threonine; (d)aspartate and glutamate; (e) glutamine and asparagine; and (f) lysine,arginine and histidine.
 11. The isolated polypeptide of claim 6, whereinthe polypeptide having at least 98% sequence identity with residues23-108 of SEQ ID NO: 2 comprises one conservative amino acidsubstitution, wherein the conservative amino acid substitution is asubstitution among amino acids selected within each of the followinggroups: (a) glycine, alanine, valine, leucine, and isoleucine; (b)phenylalanine, tyrosine, and tryptophan; (c) serine and threonine; (d)aspartate and glutamate; (e) glutamine and asparagine; and (f) lysine,arginine and histidine.
 12. An isolated polypeptide of comprising anamino acid sequence having 2 conservative amino acid substitutionswithin residues 23-108 of SEQ ID NO: 2 wherein the polypeptidestimulates gastrointestinal contractility.
 13. The isolated polypeptideof claim 12, wherein the conservative amino acid substitutions areselected from the group consisting of: a) amino acid position 35 of SEQID NO: 2 is Asp; b) amino acid position 36 of SEQ ID NO: 2 is Lys; c)amino acid position 42 of SEQ ID NO: 2 is Gly; d) amino acid position 48of SEQ ID NO: 2 is Val; e) amino acid position 50 of SEQ ID NO: 2 isIle; f) amino acid position 52 of SEQ ID NO: 2 is Val; g) amino acidposition 53 of SEQ ID NO: 2 is Lys; h) amino acid position 55 of SEQ IDNO: 2 is Ile; i) amino acid position 63 of SEQ ID NO: 2 is Lys; j) aminoacid position 66 of SEQ ID NO: 2 is Asp; k) amino acid position 71 ofSEQ ID NO: 2 is Leu; l) amino acid position 72 of SEQ ID NO: 2 is Thr;m) amino acid position 73 of SEQ ID NO: 2 is Arg; n) amino acid position80 of SEQ ID NO: 2 is Arg; o) amino acid position 93 of SEQ ID NO: 2 isAla; and p) amino acid position 102 of SEQ ID NO: 2 is Phe and whereinthe polypeptide stimulates gastrointestinal contractility.
 14. Theisolated polypeptide of claim 12, wherein the conservative amino acidsubstitutions are substitutions among amino acids selected within eachof the following groups: (a) glycine, alanine, valine, leucine, andisoleucine; (b) phenylalanine, tyrosine, and tryptophan; (c) serine andthreonine; (d) aspartate and glutamate; (e) glutamine and asparagine;and (f) lysine, arginine and histidine.
 15. An isolated polypeptide,comprising the sequence of 28 to 108 of SEQ ID NO:2.
 16. The isolatedpolypeptide of claim 15 comprising an affinity tag.
 17. The isotatedpolypeptide of claim 16, wherein the affinity tag is selected from thegroup consisting of: polyhistidine tract, protein A, glutathione Stransferase, Glu-Glu affinity tag, substance P, Flag peptide,streptavidin binding peptide, and maltose-binding protein.